Smart glass slide for microarrays

ABSTRACT

Device for use in a biosensor comprising a multisite array of test sites, the device being useful for modulating the binding interactions between a (biomolecular) probe or detection agent and an analyte of interest from a biological by modulating the pH or ionic gradient near the electrodes in such biosensor. The device provides a biosensor which is more accurate, reliable and the results of which are more reproducible. Analytic methods for more accurately measuring an analyte of interest in a biological sample are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application of U.S. patentapplication Ser. No. 13/834,126, filed on Mar. 15, 2013, the content ofwhich is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to a device for use in microarrays in a biosensorand diagnostic methods for biomolecules using such biosensor comprisingthe device. Moreover, the invention relates to a method for accuratelyand reliably controlling a pH gradient near electrode surfaces formodulating biomolecular interactions in such biosensor.

BACKGROUND

Recently there has been an increased interest in predictive,preventative, and particularly personalized medicine which requiresdiagnostic tests with higher fidelity, e.g., sensitivity andspecificity. Multiplexed measurement platforms, e.g., protein arrayscurrently used in research, are among the promising diagnosticstechnologies for the near future. The samples in these tests can behuman body fluids such as blood, serum, saliva, biological cells, urine,or other biomolecules but can also be consumables such as milk, babyfood, or water. Within this field there is a growing need for low-cost,multiplexed tests for biomolecules such as nucleic acids, proteins, andalso small molecules. Achieving the sensitivity and specificity neededin such tests is not without difficult challenges. Combining these testswith integrated electronics and using CMOS technology has providedsolutions to some of the challenges.

The two main limitations in a detection assay include sensitivity andcross-reactivity. Both of these factors affect the minimum detectableconcentration and therefore the diagnostic error rate. The sensitivityin such tests is generally limited by label detection accuracy,association factor of the probe-analyte pair (for example anantibody-antigen pair), and the effective density of probe molecule (forexample probe antibody) on the surface. Other molecules in thebiological sample can also affect the minimum detectable concentrationby binding to the probe molecule (for example the primary antibody), orby physisorption of the analyte to the surface at the test site. Thedetection agent (for example a secondary antibody) may also physisorb tothe surface causing an increase in the background signal. Solving thecross-reactivity and background problem can take a significant amount oftime in the assay development of a new test and increases the cost andcomplexity of the overall test. The assay is typically optimized byfinding the best reagents and conditions and also by manufacturing themost specific probe molecule (for example antibody). This results in along development time, the infeasibility of tests in some cases, and ahigher manufacturing cost. For example a typical development of an ELISAassay requires several scientists working for more than a year findingthe correct antibody as part of the assay development. Cross-reactivityof the proteins may be the source of the failure of such an effort.

A biosensor providing a multiple site testing platform was thought toprovide a solution to some of the above described limitations in assaydevelopment. US Published Patent Application US 2011/0091870 describessuch biosensor having multiple sites that could be subjected todifferent reaction conditions to modulate the binding of thebiomolecular analyte (for example proteins) to the probe molecule. Forexample the signal detected in a biosensor having four sites also canhave several components, e.g. four. These four terms may correspond tothe concentration of the biomarker of interest, concentration ofinterfering analytes in the sample that bind non-specifically to primaryantibody (probe molecule) sites and prevent the biomarker to bind,concentration of interfering analytes in the sample that form a sandwichand produce wrong signal, and finally the concentration of interferinganalytes in the sample that physisorb to the surface and produce wrongsignal. Each term is also proportional to a binding efficiency factor,α_(ij), which is a function of the molecule affinities and other assayconditions, e.g., mass transport. By controlling the condition at eachsite separately, different sites will have different efficiency factors.Accurate measurement of the signal at each site will result in multipleequations and multiple unknowns for example,

$\left\{ {\begin{matrix}{S_{1} = {{\alpha_{11}C_{an}} - {\alpha_{12}C_{j\; 1}} + {\alpha_{13}C_{j\; 2}} + {\alpha_{14}C_{j\; 3}}}} \\{S_{2} = {{\alpha_{21}C_{an}} - {\alpha_{22}C_{j\; 1}} + {\alpha_{23}C_{\;{j\; 2}}} + {\alpha_{24}C_{j\; 3}}}} \\{S_{3} = {{\alpha_{31}C_{an}} - {\alpha_{32}C_{j\; 1}} + {\alpha_{33}C_{j\; 2}} + {\alpha_{34}C_{j\; 3}}}} \\{S_{4} = {{\alpha_{41}C_{an}} - {\alpha_{42}C_{j\; 1}} + {\alpha_{43}C_{j\; 2}} + {\alpha_{44}C_{j\; 3}}}}\end{matrix}C_{an}} \right.$where C_(an) corresponds to the targeted biomolecular analyteconcentration and C_(j1), C_(j2), C_(j3) correspond to the totalconcentration of molecules which result in different terms in backgroundsignal.

Accurate and precise control of the assay conditions at different sitesto generate large changes in the binding efficiency factors is importantin the performance of such biosensor as a detection system for abiomolecular analyte of interest. In co-pending U.S. application Ser.No. 13/543,300 (the content of which is incorporated herein by referencein its entirety) such biosensors and such methods are described that canbe readily integrated with a CMOS, electrode array, or TFT based setupto generate large change in binding efficiencies between test sites in abiosensor having an array of multiple test sites. In order to accuratelymeasure the biomolecular analyte of interest the biosensor requires ahigh degree of reliability and reproducibility. Variations in themodulation of the local pH due to repeated use of the biosensor andvariations between subsequent measurements may decrease the accuracy ofthe determination of the biomolecular analyte of interest by suchbiosensor. As such the modulation of the pH at each site of themultisite array of the biosensor needs to be accurately controlled andvariations in such pH modulation need to be corrected. Therefore, thereis a need for a biosensor in which the pH can be accurately, reliably,and reproducibly controlled at each of the multisite array test sites.

SUMMARY OF THE INVENTION

Herein provided are devices and methods for accurately, reliably andreproducibly controlling the pH that can be integrated with for examplea CMOS, electrode array, or TFT based biosensor having an array ofmultiple test sites. In particular, the current application providesmethods to reliably and reproducibly modulate the pH or ionicconcentration near electrode surfaces of such biosensors in order tomodulate the biomolecular interactions between a probe biomolecule and abiomolecular analyte of interest. The device described herein can beused in a biosensor as described in co-pending application Ser. No.13/543,300 in order to repeatedly determine a biomolecular analyte ofinterest in a sample while maintaining a high degree of accuracy of thebiosensor.

In one embodiment there is provided a device for use in a biosensorhaving a multisite array of test sites, the device comprising:

-   -   (a) a transparent support substrate supporting one or more        electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.

In another embodiment there is provided biosensor comprising the devicecomprising:

-   -   (a) a transparent support substrate supporting one or more        electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.

In another embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a transparent support substrate supporting one        or more electrodes, and a biomolecular interface layer having        immobilized pH sensitive Fluorescent Protein and one or more        immobilized probes thereon; and    -   b) reacting at the one or more electrodes an electrochemically        active agent in an aqueous solution to produce H+ ion or OH−        ions.

In yet another embodiment there is provided a method for detecting abiomolecule analyte in a biological sample, the method comprising:

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a transparent support substrate supporting one        or more electrodes and a biomolecular interface layer having        immobilized pH sensitive Fluorescent Protein and one or more        immobilized probes thereon, and at each test site having an        aqueous solution comprising a dilute phosphate buffer and an        electrochemically active agent;    -   b) at each test site electrochemically reacting the        electrochemically active agent in an aqueous solution to produce        H+ ion or OH− ions, thereby modulating and controlling the pH at        each test site;    -   c) adding a biological sample to each test site; and    -   d) detecting the biomolecule analyte in each test site,        wherein the amounts of electrochemically active agent and the        electrochemical reaction are varied between test sites in a        subset array of test sites in order to obtain sets of test sites        in which the pH or ionic concentration near electrode surfaces        in the test sites varies, and wherein the pH at each test site        is determined by the fluorescence intensity of the pH sensitive        Fluorescent Protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Illustration of a substrate (glass or plastic) (1) with an arrayof electrodes (2) onto which a biomolecular interface layer (10) isapplied which include fluorescence protein (such as Green FluorescenceProtein (GFP)) spots (5), and immobilized probes (4), immobilized usinga polyethylene glycol (PEG) linker (3).

FIG. 2: shows the change in the fluorescence intensity of GFP covalentlybound to the PEG-coated ITO in response to the change in solution pH.The solution pH was adjusted by adding HCl to a dilute phosphate buffer(pH 7.4).

FIG. 3: shows the pH change at the surface of ITO working electrodegenerated via current-driven oxidation of a redox active molecule,2-methyl-1,4-dihydroquinone, in diluted phosphate buffer (pH=7.4)containing 0.1M Na₂SO₄. After 10 seconds of induction, current (50microamps) was applied for 30 second, which resulted in a drop ofsolution pH to 5.5, as was observed by a change in GFP fluorescenceintensity (FIG. 2 is used as calibration curve to assess the pH values).After current was turned off, the pH recovered to neutral value within50 seconds.

FIG. 4: illustrates the visual changes in the GFP spot before, duringand after pH modulation experiment. A, the profile of fluorescenceintensity across the spot is shown. B, the changes in the GFP spotfluorescence intensity are shown before (0 sec), during (40 sec), andafter (110 sec) applying a current through an electrode.

FIG. 5: shows a glass slide with an ASIC chip interfaced to transparentITO electrodes.

DETAILED DESCRIPTION

Methods to modulate the pH in a biosensor having a multisite array oftest sites are described in co-pending U.S. patent application Ser. No.13/543,300. When used in a biosensor the accuracy, reliability andreproducibility of the modulation of the pH at each test site isimportant. However the modulation of the pH at each test site may varybetween subsequent uses. In order to accurately determine the amount ofa biomolecular analyte of interest in a sample using the biosensor andmethod described in the aforementioned co-pending U.S. patentapplication Ser. No. 13/543,300 the pH at each test site needs to beaccurately modulated or controlled. The device provided herein allowsfor accurate determination and control of the pH at each test site insuch biosensor, the device comprising:

-   -   (a) a transparent support substrate supporting one or more        electrodes; and    -   (b) a biomolecular interface layer having immobilized pH        sensitive Fluorescent Protein and one or more immobilized probes        thereon.        The transparent support substrate in the device described herein        is preferably a glass or plastic substrate but also be any other        transparent non-glass substrate.

The immobilized pH sensitive fluorescent protein allows for sensing thepH at an electrode once the electrode (working electrode) causesmodulation of the pH at a particular test site such as a test site in amultisite array. The fluorescence intensity of the fluorescent proteinchanges due to modulation of the pH. The change in fluorescenceintensity of the fluorescent protein is proportional to the change inthe pH (there is a linear relationship between the pH and thefluorescence intensity). Therefore, as is also shown in FIG. 2, the pHvalue at each location at any time when the biosensor is in use can bereadily obtained by correlating the fluorescence intensity of thefluorescent protein with the pH. An accurate calibration of thecorrelation between pH and fluorescence intensity may be carried outbefore or during use of the biosensor. When the calibration is carriedout during use of the biosensor one or more test sites within amultisite array may be dedicated to calibration of the fluorescenceintensity to pH correlation. When the pH is no longer modulated at suchtest site by the electrode (working electrode) the fluorescenceintensity of the immobilized fluorescent protein reverts back to itsintensity before a current was applied through the electrode.

Preferably, the immobilized fluorescent protein is selected from animmobilized green fluorescent protein, an immobilized yellow fluorescentprotein, and an immobilized cyan fluorescent protein. More preferably,the immobilized fluorescent protein is immobilized Green FluorescentProtein (GFP). In an alternative embodiment immobilized pH sensitivedyes may be used on the transparent support substrate instead of animmobilized pH sensitive fluorescent protein. In a multisite array oftest sites in a biosensor the immobilized fluorescent protein covers onthe substrate an area that is also covered by an electrode and an areathat is not covered with an electrode. The electrode covered by theimmobilized fluorescent protein is either a working electrode or acounter electrode. Preferably, the immobilized fluorescent protein isapplied onto the substrate as distinct spots, wherein each spot overlapswith only one test site and an area not covered by an electrode as shownin FIG. 4A. The presence of fluorescent protein on an area that is notcovered by an electrode allows for the determination, within thebiosensor, of fluorescence intensity when the pH is not modulated by theelectrode. This fluorescence intensity can be used as a standard andcontrol in determining whether, after ceasing modulation of the pH by anelectrode the fluorescence intensity will revert back to its originalintensity. Accordingly, in a method for detecting a biomolecular analytein a biological using the device, the fluorescent protein not located onor near an electrode can be used as an internal reference for signalnormalization.

The device includes one or more counter electrodes and one or moreworking electrodes. In the device one or more electrodes can be arrangedin a multisite array, each site of the multisite array comprising aworking electrode and/or counter electrode. The electrodes can be anyelectrode suitable in a biosensor for example indium tin oxide (ITO),gold, or silver electrodes. In a preferred embodiment the electrodes inthe device are indium tin oxide (ITO) electrodes. In an alternativeembodiment the working electrode is an indium tin oxide electrode andthe counter electrode(s) is selected from an indium oxide electrode, agold electrode, a platinum electrode, a silver electrode, and a carbonelectrode.

The electrodes in the device may be used either for modulating the pH oras sensing electrodes or both. In the device or biosensor using thedevice, the one or more electrodes are connected to an electronic boardvia pogo-pins, a chip on foil via z-axis adhesive, or a chip on thesubstrate. The electronic board or chip are powered by a printedbattery, a small battery bound to the substrate, a magnetically coupledpower transfer using coils on the substrate, or a rf-coupled powertransfer using coils on the substrate.

The biomolecular probe is attached or immobilized onto the supportand/or electrode(s) within a biomolecular interface layer. Thebiomolecular layer includes a layer of immobilized polymers, preferablya silane immobilized polyethylene glycol (PEG). Surface-immobilizedpolyethylene glycol (PEG) can be used to prevent non-specific adsorptionof biomolecular analytes onto surfaces. At least a portion of thesurface-immobilized PEG can comprise terminal functional groups such asN-hydroxysuccinimide (NHS) ester, maleimide, alkynes, azides,streptavidin or biotin that are capable of conjugating. The biomolecularprobe may be immobilized by conjugating with the surface-immobilizedPEG. It is important that during operation of the device the change ofthe pH does not impair the covalent binding of for example the PEG ontothe surface of a solid support, or the linker that conjugated thebiomolecular probe to the PEG.

A suitable biomolecular probe can be a carbohydrate, a protein, aglycoprotein, a glycoconjugate, a nucleic acid, a cell, or a ligand forwhich the analyte of interest has a specific affinity. Such probe canfor example be an antibody, an antibody fragment, a peptide, anoligonucleotide, a DNA oligonucleotide, a RNA oligonucleotide, a lipid,a lectin that binds with glycoproteins and glycolipids on the surface ofa cell, a sugar, an agonist, or antagonist. In a specific example, thebiomolecular probe is a protein antibody which interacts with an antigenthat is present for example in a biological sample, the antigen being abiomolecular analyte of interest.

A biosensor comprising the device provided herein can be used in ananalytical method for determining a biomolecular analyte of interest ina biological sample, which can be for example a protein, such as anantigen or enzyme or peptide, a whole cell, components of a cellmembrane, a nucleic acid, such as DNA or RNA, or a DNA oligonucleotide,or a RNA oligonucleotide.

In such method a local pH or ionic concentration gradient can beobtained at various test sites in a multisite array biosensor. Thevariation of the local pH and/or ionic concentration gradient at theelectrode, and in particular in the vicinity of the (biomolecular) probein a biomolecular interface layer, over subsets of the multisite arrayof the biosensor, allows for modulating the binding efficiency of the(biomolecular) probe and an analyte to be tested from a biologicalsample. The analyte of interest, when bound to the (biomolecular) probe,can be then detected using a detection agent, such as for example alabeled secondary antibody. The modulation of binding efficiencies in asubset of a multisite array provides a method for the accuratedetermination of such analyte of interest.

The device preferably a multisite array of test sites within abiosensor, which multisite array is for example described in US2011/0091870. Such multisite array preferably includes a number ofdifferent subarrays/subsets of test sites. Each test sites represents asite for performing an analysis of a (biomolecular) analyte from abiological sample through the detection of the (biomolecular) analyteusing a (biomolecular) probe. The analytical conditions in each testsite in each of the subarrays/subsets may be varied to obtain acollection of varied signals that will result in multiple equations andmultiple unknowns from which the concentration of the (biomolecular)analyte can be determined in order to obtain an accurate measurement ofthe (biomolecular) analyte.

The multiple unknowns in the obtained varied signals each includes aterm that is proportional to a binding efficiency factor, α_(ij), andthe concentrations of the various molecules in the biological samplebinding that are detected at the test site. The multiple equations withmultiple unknowns may be represented for example as follows,

$\left\{ {\begin{matrix}{S_{1} = {{\alpha_{11}C_{an}} - {\alpha_{12}C_{j\; 1}} + {\alpha_{13}C_{j\; 2}} + {\alpha_{14}C_{j\; 3}}}} \\{S_{2} = {{\alpha_{21}C_{an}} - {\alpha_{22}C_{j\; 1}} + {\alpha_{23}C_{\;{j\; 2}}} + {\alpha_{24}C_{j\; 3}}}} \\{S_{3} = {{\alpha_{31}C_{an}} - {\alpha_{32}C_{j\; 1}} + {\alpha_{33}C_{j\; 2}} + {\alpha_{34}C_{j\; 3}}}} \\{S_{4} = {{\alpha_{41}C_{an}} - {\alpha_{42}C_{j\; 1}} + {\alpha_{43}C_{j\; 2}} + {\alpha_{44}C_{j\; 3}}}}\end{matrix}C_{an}} \right.$where C_(an) corresponds to the targeted biomolecular analyteconcentration and C_(j1), C_(j2), C_(j3) correspond to the totalconcentration of molecules which result in different terms in backgroundsignal, from which collection of multiple equations the concentration ofthe targeted biomolecular analyte can be determined.

The number of subarrays/subsets, as well as the number of test siteswithin each subarray/subset may be varied, as needed to obtain suchaccurate measurement of the analyte. Some of these analytical conditionsinclude parameters such as for example temperature, shear stress, andpressure. For example the temperature of the aqueous solution in whichthe biomolecular probe and analyte of interest in the biological sampleinteract can be varied using the electromagnetic heat at the test site.Another important condition for the interaction between the biomolecularprobe and the analyte of interest is the pH or ionic concentration.

The device provided herein and used in a biosensor comprises such arrayof multiple test sites in solution in order to modulate the pH at eachtest site and to determine the presence and concentration of abiomolecular analyte of interest in a biological sample. In such use thedevice is in contact with an aqueous solution comprising a phosphatebuffer, preferably a diluted phosphate buffer which preferably has aconcentration of 0.1 mM to 100 mM. In a preferred embodiment the pH ofthe diluted phosphate buffer is between 5 and 8, preferably between 7and 8, and more preferably between 7 and 7.5.

The aqueous solution may further comprise one or more additionalelectrolytes, such as for example sodium sulfate, or any other suitablestrong electrolyte. Preferably, the additional electrolyte is selectedfrom sodium sulfate, sodium or potassium chloride, sodium or potassiumbromide, sodium or potassium iodide, sodium or potassium perchlorate,sodium or potassium nitrate, tetraalkylammonium bromide andtetraalkylammonium iodide. Buffer-inhibitors may also be used in theaqueous solution. Suitable buffer inhibitors may be selected frompoly(allylamine hydrochloride), poly (diallyldimethyl ammoniumchloride), poly(vinylpyrrolidone), poly(ethyleneimine),poly(vinylamine), poly(4-vinylpyridine) andtris(2-carboxyethyl)phosphine hydrochloride. When used in a method tomodulate the pH such as described in co-pending U.S. patent applicationSer. No. 13/543,300 the aqueous solution preferably also comprises awater-miscible organic co-solvent selected from the groups consisting ofacetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),N,N-dimethyl acetamide (DMAc), and mixtures thereof.

In such method the pH modulation on the device provided herein can becarried out using an electrochemically active agent. Suitableelectrochemically active agents include dopamine hydrochloride, ascorbicacid, phenol and derivatives, benzoquinones and derivatives, forexample, 2,5-dihydroxy-1,4-benzoquinone,2,3,5,6-tetrahydroxy-1,4-benzoquinone and2,6-dichloroquinone-4-chloroimide; naphthoquinones and derivatives, forexample, hydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone,and potassium 1,4-naphthoquinone-2-sulfonate; and 9,10-anthraquinone andderivatives, for example, sodium anthraquinone-2-carboxylate, potassium9,10-anthraquinone-2,6-disulfonate. Preferably the concentration of theelectrochemically active agent in the aqueous solution is from 1 nM to100 mM.

In another embodiment is provided a method of modulating the pH usingthe device in a biosensor. The method of modulating the pH or ionicconcentration in a biosensor comprises:

-   -   a) providing a biosensor including one or more devices as        described herein comprising a multisite array of test sites in        which the conditions for interacting with a biomolecule analyte        can be varied independently; and    -   b) reacting at the one or more electrodes an electrochemically        active agent in an aqueous solution to produce H+ ion or OH−        ions.        In the method the concentration of the electrochemically active        agent in the aqueous solution is preferably from 1 nM to 100 mM.

In a method of modulating the pH in a biosensor using the devicedescribed herein the electrochemically active agent may beelectro-oxidized or electro-reduced at an electrode potential in therange of −2V to +2V. Preferably the electrode potential is in the rangeof −1V to +1V, even more preferably the electrode potential is in therange of −0.5V to +0.5V. The voltage required to drive the redoxreaction can be used as a real time feedback method to monitor pH thatis produced at the electrode surface.

Modulation of the pH or ionic concentration on a device in a biosensordescribed herein by electrochemical reaction at the one or moreelectrode may be carried out in a galvanostatic mode or potentiostaticmode. In addition, any type of electrical pulse may be applied on theelectrodes of the device in the method for modulating the pH. Such pulsemay be in the form of an annealing pulse and may vary by pulsefrequency, pulse width, and pulse shape. In an annealing pulse asufficient voltage is applied to change the pH to such that non-coventlybound molecules from the biological sample are removed from the devicein the biosensor. Such annealing pulse eliminates or reduces the needfor washing the substrate following first contact with a sample in orderto remove non-covalently bound material. Another advantage is that theannealing pulse may be more efficient to remove such non-covalentlybound material from the device than a simple washing. A preferred pulsewidth for modulating the pH is in the range of 1 nanosecond to 60minutes.

In another embodiment there is provided an analytical method of usingthe device described herein in a biosensor to determine the presenceand/or concentration of a biomolecular analyte of interest in abiological sample. This analytical method comprises

-   -   a) providing a biosensor comprising a multisite array of test        sites in which the conditions for interacting with a biomolecule        analyte can be varied independently, the biosensor having a        device comprising a transparent support substrate supporting one        or more electrodes and a biomolecular interface layer having        immobilized pH sensitive Fluorescent Protein and one or more        immobilized probes thereon, and at each test site having an        aqueous solution comprising a dilute phosphate buffer and an        electrochemically active agent;    -   b) at each test site electrochemically reacting the        electrochemically active agent in an aqueous solution to produce        H+ ion or OH− ions, thereby modulating and controlling the pH at        each test site;    -   c) adding a biological sample to each test site; and    -   d) detecting the biomolecule analyte in each test site,        wherein the amounts of electrochemically active agent and the        electrochemical reaction are varied between test sites in a        subset array of test sites in order to obtain sets of test sites        in which the pH or ionic concentration near electrode surfaces        in the test sites varies, and wherein the pH at each test site        is determined by the fluorescence intensity of the pH sensitive        Fluorescent Protein.

The biomolecular analyte can be detected using any suitable detectionmethod. Known detection methods of such analyte include luminescence,fluorescence, colorimetric methods, electrochemical methods, impedancemeasurements, or magnetic induction measurements. In various of suchmethods the analyte binds to the immobilized biomolecular probe and adetection agent such as for example a secondary labeled probe thatspecifically binds to the analyte, bound to the immobilized probe, isintroduced. This detection agent or secondary labeled probe gives riseto a detectable signal such as for example luminescence or fluorescence.

The following description is an illustration of a specific embodimentwhich may be modified within the scope of the description as would beunderstood from the prevailing knowledge. FIG. 1, shows a side view of apart of the device which includes a substrate (1) from glass or plastic.One or more electrodes (2) are covered onto the substrate (1) which isalso covered with a biomolecular interface layer (10). The biomolecularinterface layer (10) comprises immobilized PEG (3), immobilized probe(4) and immobilized pH sensitive fluorescent protein in the form ofGreen Fluorescent Protein spots (5). The GFP spots (5) overlap with anelectrode (2) and an area that is not covered by an electrode. Theelectrodes (2) and the GFP spots (5) are arranged in a multisite arrayso as to provide multiple test sites on the device.

The following are examples which illustrate specific methods without theintention to be limiting in any manner. The examples may be modifiedwithin the scope of the description as would be understood from theprevailing knowledge.

Examples

Electrochemical Modulation of pH as Monitored by Fluorescence Intensitywith Green Fluorescence Protein (GFP)

Electrode material used: The electrode material was indium tin oxide.The fluorescent protein used is GFP immobilized on a glass substratewhich includes an array of electrodes. The GFP is applied as spots, eachspot covers an area that overlaps with one electrode and an area that isnot overlapping with an electrode.

The pH change at the surface of ITO working electrode is generated viacurrent-driven oxidation of a redox active molecule,2-methyl-1,4-dihydroquinone, in diluted phosphate buffer (pH=7.4)containing 0.1M Na₂SO₄. After 10 seconds of induction, current (50microamps) was applied for 30 second, which resulted in a drop ofsolution pH to 5.5, as was observed by a change in GFP fluorescenceintensity. FIG. 2 is used as calibration curve to assess the pH values.After current was turned off, the pH recovered to neutral value within50 seconds (as shown in FIGS. 3 and 4B).

What is claimed is:
 1. A method for detecting a biomolecule analyte in abiological sample, the method comprising: (a) adding a dilute phosphatebuffer and an electrochemically active agent to an aqueous solution ofeach test site in a multisite array of test sites in a biosensor,wherein conditions for interacting with the biomolecule analyte at eachof the test sites can be varied independently; (b) electrochemicallyreacting the electrochemically active agent in the aqueous solution ateach test site through one or more electrodes supported on a glass orplastic support substrate to produce hydrogen ions (H⁺) or hydroxideions (OH⁻), thereby modulating and controlling the pH at each test site;(c) adding a biological sample to the aqueous solution of each of thetest sites, wherein: each of the test sites includes a biomolecularinterface layer that includes: (i) a layer of immobilized polymer thatcovers the glass or plastic support substrate; (ii) one or more pHsensitive fluorescent proteins immobilized by attachment directly to thelayer of immobilized polymer; and (iii) one or more probes immobilizedby attachment directly to the layer of immobilized polymer; a firstportion of the one or more pH sensitive fluorescent proteins is locatedwithin a maximum distance from the one or more electrodes within whichthe one or more electrodes is able to affect pH of the one or more pHsensitive fluorescent proteins; and a second portion of the one or morepH sensitive fluorescent proteins is located more than the maximumdistance from the one or more electrodes; (d) determining the pH at eachof the test sites using a fluorescence intensity of the one or more pHsensitive fluorescent proteins; and (e) detecting the biomoleculeanalyte in each of the test sites; wherein: amounts of theelectrochemically active agent and the electrochemical reaction arevaried between the test sites in a subset of the array of test sites inorder to obtain sets of test sites in which the pH or ionicconcentration at the one or more electrode surfaces in the test sitesvaries to produce a pH or ionic concentration gradient at the one ormore electrodes and in a vicinity of the one or more probes in thebiomolecular interface layer; and the one or more pH sensitivefluorescent proteins is one or more Green Fluorescent Proteins.
 2. Themethod of claim 1, wherein the second portion of the one or more pHsensitive fluorescent proteins is used as an internal reference forsignal normalization.